Dexamethasone Inhibits Tumor Necrosis Factor-a ... - Semantic Scholar

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Dexamethasone Inhibits Tumor Necrosis Factor-a-stimulated Gastric Epithelial Cell Migration Jiing-Chyuan Luo1,2*, Chi-Hin Cho3, Ka-Man Ng1, Kuo-Wei Hsiang1, Ching-Liang Lu1,2, Tseng-Shing Chen1,2, Full-Young Chang1,2, Han-Chieh Lin1,2, Chin-Lin Perng1,2, Shou-Dong Lee1,2 1

Division of Gastroenterology, Department of Medicine, Taipei Veterans General Hospital, Department of Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan, R.O.C., and 3 Department of Pharmacology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China. 2

Background: Cell migration (restitution) occurs in the early phase of gastric ulcer healing. Tumor necrosis factor (TNF)-α is overexpressed at the ulcer margin and plays a physiologic role in gastric ulcer healing. Dexamethasone, which is a potent corticosteroid, delays rat gastric ulcer healing. We evaluated whether dexamethasone inhibited TNF-α-stimulated gastric epithelial cell migration using a rat normal gastric epithelial cell line (RGM-1). Methods: An artificial wound model was employed to measure cell migration. Western blot was performed to evaluate the possible mechanisms. Intracellular prostaglandin E2 level was measured using an enzyme-linked immunosorbent assay. Results: TNF-α treatment (10 ng/mL) for 12–48 hours significantly increased RGM-1 cell migration, and TNF-α treatment increased cyclooxygenase (COX)-2 protein expression 8 hours later and prostaglandin E2 (PGE2) synthesis 12 hours later compared with control (p < 0.05). Dexamethasone (10–6 M) significantly inhibited the stimulatory effect of TNF-α on RGM-1 cell migration, which was associated with a significant decrease in COX-2 expression and PGE2 level in cells (p < 0.05). Conclusion: TNF-α plays a regulatory role in rat gastric epithelial cell migration and dexamethasone inhibited TNF-αstimulated cell migration, which was associated with a decrease in COX-2 expression and PGE2 formation. [J Chin Med Assoc 2009;72(10):509–514] Key Words: cell migration (restitution), cyclooxygenase-2, dexamethasone, gastric ulcer healing, tumor necrosis factor-α

Introduction Ulcer formation is a dynamic imbalance between mucosal aggressive factors and defensive factors. When the function of defense and repairing factors is less than that of aggressive factors, mucosal injury worsens, and finally ulcer formation develops. Ulcer healing and repair requires a reconstruction of the surface epithelium and underlying connective tissue, including blood vessels and muscle layers.1 The early phase of mucosal repair occurs in the absence of cellular proliferation and is termed restitution or cell migration.2,3 The late phase of repair of deeper tissue damage involves cell proliferation as well as angiogenesis of granulation tissue to reconstruct the mucosal and submucosal

architecture.1,4 All of these events are controlled and regulated by cytokines, growth factors, and some transcription factors that are overexpressed or activated over injured mucosa or ulcer margins.5–7 In general, re-epithelialization, the migration of epithelial cells from the wound margin to restore epithelial continuity, is essentially an early process for gastrointestinal ulcer healing.2,8,9 Cell migration requires cytoskeletal rearrangement: actin microfilament assembly, stress fiber and lamelipodia formation, as well as rearrangement of focal adhesion complex attachments to the extracellular matrix.10 Growth factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF), as well as cytokines such as tumor necrosis

*Correspondence to: Dr Jiing-Chyuan Luo, Division of Gastroenterology, Department of Medicine, Taipei Veterans General Hospital, 201, Section 2, Shih-Pai Road, Taipei 112, Taiwan, R.O.C. E-mail: [email protected] Received: May 20, 2009 Accepted: September 7, 2009 ●

J Chin Med Assoc • October 2009 • Vol 72 • No 10 © 2009 Elsevier. All rights reserved.

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factor (TNF)-α, interleukin (IL)-1β and IL-8, are reported to activate cell migration.2,7 The expression of TNF-α is increased at ulcer sites in rat stomachs; TNF-α is presumably secreted from macrophages and mononuclear cells.2,11 TNF-α has been reported to indirectly stimulate cell migration, partially through the increased production of HGF and IL-8.2,12 Our previous in vivo studies demonstrated that dexamethasone, which is a potent corticosteroid, delayed rat gastric ulcer healing by inhibiting epithelial cell proliferation and angiogenesis at the ulcer margin.1,4 Our previous in vitro study also showed that dexamethasone inhibited TNF-α-stimulated gastric epithelial cell proliferation via inhibition of arachidonic acid– cyclooxygenase (COX)-2 pathway activation.13 In this in vitro study, we evaluated whether dexamethasone inhibited TNF-α-stimulated gastric epithelial cell migration and explored the possible mechanistic pathways. The findings further explained the mechanisms of how glucocorticoids delay gastric ulcer healing.

Methods All study chemicals were purchased from Sigma (Sigma Chemical Co., St Louis, MO, USA) unless otherwise specified. Ten μg TNF-α (human, recombinant) was prepared in 2 mL of 0.01 M phosphate buffer saline at pH 7.4 containing 0.1% bovine serum albumin as a stock solution.

Cell culture The RGM-1 rat gastric mucosal cells were established from normal Wistar rats and were a gift from Dr Hirofumi Matsui of Tsukuba University (RCB0876 at Riken Cell Bank, Tsukuba, Japan). RGM-1 cells are epithelial in origin and like mucous epithelial or mucous neck cells.14,15 Ours were grown in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 100 U/mL penicillin G, 100 μg/mL streptomycin, and 20% fetal bovine serum (FBS) (Gibco BRL) in an incubator at 37°C and 5% carbon dioxide.

duration of mitomycin almost abolished 10% of FBSinduced RGM-1 cell proliferation as reflected by a 96% decrease in BrdU (5-bromo-2⬘-deoxy-uridine) labeling compared to cells incubated with medium alone at 48 hours.10 An artificial circular wound of cell-free area 2 mm2 was made in the center of the monolayer using a plastic blade.16,18 The wounded monolayer was then cultured in the medium with 1% FBS in the presence of control, or TNF-α alone (2 and 10 ng/mL), or TNF-α (10 ng/mL) with dexamethasone (10–8 M and 10–6 M), or dexamethasone alone (10–8 M and 10–6 M). The size of the cell-free area was monitored from time 0 to 48 hours using a digital image processor connected to a microscope (Nikon, Tokyo, Japan).18 The areas were calculated with an image-analyzing program (Leica, Cambridge, England).

Western blot analysis for COX-1, COX-2, EGF, and bFGF After synchronization and pretreatment with mitomycin C as described above, cells were treated with DMEM/F-12 containing 1% FBS and 10 ng/mL TNF-α in the presence or absence of 10–6 M dexamethasone, or 10–6 M dexamethasone alone for 8 hours. Cells were then collected in radioimmunoprecipitation assay buffer for Western blot analysis. Following sonication and centrifugation, protein concentration was measured using a protein assay kit (BioRad Laboratories, Hercules, CA, USA). Proteins were separated by SDS-polyacrylamide electrophoresis gel overlaid with a 10% acrylamide stacking gel, and then transferred to Hybond C nitrocellulose membranes (Amersham International Plc., Amersham, UK). Membranes were probed with antibodies against COX-1 (1:800), COX-2 (1:500), EGF (1:500), bFGF (1:500), and β-actin (1:2,000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight at 4°C and incubated for 1 hour with secondary antibodies conjugated with peroxidase. The membrane was developed using an enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ, USA) and was exposed to X-ray film (Fuji Photo Film, Tokyo, Japan). Quantitation was performed using a densitometer (Scan Marker III; Microtek, Carson, NV, USA).

Cell migration Cells were seeded in 24-well culture plates and cultured in DMEM/F-12 with 20% FBS until confluence. After confluence, monolayers of the cells were starved for 24 hours in the medium containing 1% FBS. The cells were then pretreated with mitomycin C (2 μg/mL) for 2 hours before a wound was made to inhibit cell proliferation.16,17 This dose and treatment

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Measurement of PGE2 level After synchronization and pretreatment with mitomycin C as described above, cells were treated with 10 ng/mL TNF-α in the presence or absence of dexamethasone (10–8 M and 10–6 M), or dexamethasone (10–6 M) alone for 12 hours. Then, cells were homogenized with homogenizing buffer (0.05 M Tris-HCl

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Statistical analysis Analytic results are expressed as mean ± standard deviation. There were six samples in each group. Differences between the means were analyzed with the Mann-Whitney U test when appropriate. Bonferroni correction was performed to adjust for the fact that multiple comparisons were done in each experiment. A p value < 0.05 was considered to be statistically significant.

Results Effects of TNF-a on cell migration in RGM-1 cells Treatment with TNF-α 2 ng/mL increased RGM-1 cell migration, but there was no significant difference when compared with control after 12, 24, 36 and 48 hours of treatment. However, treatment with TNF-α 10 ng/mL for 12, 24, 36 and 48 hours significantly increased cell migration when compared with control (Figure 1). Thereafter, we chose 10 ng/mL TNF-α as the working concentration for subsequent experiments in this study.

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Figure 1. Effect of TNF-α on cell migration in RGM-1 cells. Cells were incubated with control or TNF-α (2 ng/mL and 10 ng/mL) for 48 hours. Sizes of the cell-free area were monitored at 0, 12, 24, 36 and 48 hours using a digital image processor connected to a microscope. The areas were calculated with an image-analyzing program. Values are mean ± standard deviation for six samples per group. *p < 0.05 vs. control group.

Cell-free area (mm2) (% of control)

at pH 7.4, 0.1 M NaCl, 0.001 M CaCl2, 1 mg/mL D-glucose, 28 μM indomethacin to inhibit further PGE2 formation) for 30 seconds.15 After centrifugation at 24,148g for 15 minutes at 4°C, the supernatants were assayed using a commercially available PGE2 enzyme-linked immunosorbent assay (Quantikine; R&D Systems Inc., Minneapolis, MN, USA). The assay procedures were in accordance with the protocol suggested in the kit. Optical densities were determined with the MRX microplate reader (Dynex Technologies Inc., Chantilly, VA, USA) at 405 nm. The amount of protein in the sample was determined by a protein assay kit, and the intracellular PGE2 level was expressed as pg/mg protein.15


Steroid-inhibited epithelial cell migration

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Figure 2. Effect of dexamethasone on cell migration in RGM-1 cells. Cells were incubated with control or dexamethasone (10–8 M and 10–6 M) for 48 hours. Sizes of the cell-free area were monitored at 0, 12, 24, 36 and 48 hours using a digital image processor connected to a microscope. Values are mean ± standard deviation of six samples per group.

Effects of dexamethasone on cell migration in RGM-1 cells

no significant difference between the TNF-α plus dexamethasone group and the control group (Figure 3).

Dexamethasone treatment alone (10–8 M and 10–6 M) for 12, 24, 36 and 48 hours did not have a stimulatory or inhibitory action on RGM-1 cell migration when compared with that of control (Figure 2).

Effects of TNF-a and dexamethasone on protein expression of COX-1, COX-2, EGF and bFGF

Effects of dexamethasone on TNF-a stimulated RGM-1 cell migration Dexamethasone treatment (10–8 M and 10–6 M) for 24, 36 and 48 hours significantly inhibited TNF-α (10 ng/mL) stimulated RGM-1 cell migration when compared with the TNF-α-treated group. There was


Treatment with TNF-α 10 ng/mL for 8 hours significantly increased COX-2 expression when compared with that of the control group. In contrast, dexamethasone (10–6 M) alone did not decrease COX-2 expression when compared with the control group (Figure 4). However, the same concentrations of dexamethasone (10–6 M) significantly decreased TNF-αstimulated COX-2 expression when compared with

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Control TNF-α 10 ng/mL TNF-α 10 ng/mL + Dexamethasone 10−8 M TNF-α 10 ng/mL + Dexamethasone 10−6 M Figure 3. Effects of dexamethasone on TNF-α stimulated RGM-1 cell migration. Cells were incubated with control, TNF-α (10 ng/mL), or TNF-α (10 ng/mL) plus dexamethasone (10–8 M or 10–6 M) for 48 hours. Sizes of the cell-free area were monitored at 0, 12, 24, 36 and 48 hours using a digital image processor connected to a microscope. Values are mean ± standard deviation for six samples per group. *p < 0.05 vs. control group; †p < 0.05 vs. TNF-α group.

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Control TNF-α 10 ng/mL TNF-α 10 ng/mL + Dexamethasone 10−8 M TNF-α 10 ng/mL + Dexamethasone 10−6 M Dexamethasone 10−6 M Figure 5. Effects of TNF-α and dexamethasone on intracellular PGE2 level. Cells were incubated with TNF-α 10 ng/mL in the absence or presence of dexamethasone (10–8 M and 10–6 M) and dexamethasone 10–6 M alone for 12 hours and measured by enzyme-linked immunosorbent assay. Values are mean ± standard deviation of six samples per group. *p < 0.05 vs. control group; † p < 0.05 vs. TNF-α treated group.

the TNF-α-treated group (Figure 4). There were no significant differences in the expressions of COX-1, EGF and bFGF among the different treatment groups, including the control, TNF-α alone, dexamethasone alone, and TNF-α plus dexamethasone treated groups (data not shown).

Effects of TNF-a and dexamethasone on intracellular PGE2 level

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Treatment with TNF-α 10 ng/mL for 12 hours significantly increased intracellular PGE2 level when compared with that of the control group (Figure 5). Again, dexamethasone treatment (10–8 M and 10–6 M) decreased PGE2 level in the TNF-α-treated group and reached a significant difference in the higher concentration group (10–6 M dexamethasone) (Figure 5).

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Figure 4. Effect of TNF-α on the protein expression of COX-2 in RGM-1 cells. Cells were incubated with TNF-α 10 ng/mL in the absence or presence of dexamethasone (10–8 M and 10–6 M) and dexamethasone (10–6 M) alone for 8 hours and measured by Western blot method. Values are mean ± standard deviation of six samples per group. *p < 0.05 vs. control group; †p < 0.05 vs. TNF-α treated group.

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Discussion Our study demonstrated that TNF-α increased COX-2 expression and PGE2 formation, and stimulated rat gastric epithelial (RGM-1) cell migration. Dexamethasone inhibited TNF-α-stimulated RGM-1 cell migration, which was associated with a decrease in COX-2 expression and PGE2 formation. These findings further explain the reasons and mechanisms for how glucocorticoids delay gastric ulcer healing. Our previous study showed that TNF-α (2 and 10 ng/mL) and dexamethasone (10–8 M and 10–6 M)


Steroid-inhibited epithelial cell migration

alone did not alter cell viability (by MTT [3-(4,5dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide] reduction method) or induce RGM-1 cell apoptosis (by DNA laddering), respectively.13 That study also showed that TNF-α, which was overexpressed in the gastric ulcer margin,11 played a positive role in RGM-1 cell proliferation via activating the arachidonic acid–COX-2 pathway, which is important for mucosal defense and ulcer healing.1,13 The current study showed that TNF-α at the same concentration (10 ng/mL) significantly stimulated COX-2 expression and PGE2 formation, and increased RGM-1 cell migration, which is the initial step in mucosal repair before cellular proliferation. In fact, activation of the COX-2 pathway is essential for cell migration.19,20 TNF-α also upregulated the production of HGF and IL-8, which promoted cell migration.2,12,20 This study also revealed that dexamethasone inhibited TNF-α-stimulated COX-2 expression and PGE2 formation, and inhibited TNF-α-stimulated RGM-1 cell migration. It was interesting to find that dexamethasone alone did not have an inhibitory action on basal RGM-1 cell migration when compared with that of control, but did have an inhibitory action on TNF-α-stimulated cell migration when compared with the TNF-α-treated group. The findings are in agreement with data showing that COX-2 expression and intracellular PGE2 levels were comparable between the control group and the dexamethasone (10–6 M) group, although COX-2 expression and PGE2 synthesis were a little weaker in the dexamethasone group than in the control group. Further study is needed to clarify why dexamethasone alone did not have an inhibitory action on basal RGM-1 cell migration. The concentrations of dexamethasone (10–8 M and 10–6 M) we used in this study are similar to the pharmacologic concentration that is found in the plasma of patients treated with dexamethasone.20,21 Putting together the findings of our previous studies and the current study, we demonstrated that dexamethasone inhibited EGF, bFGF, TNF-α-stimulated RGM-1 cell proliferation, and TNF-α-stimulated RGM1 cell migration.13,15,22 We also found that dexamethasone inhibited EGF, bFGF, and TNF-α-stimulated COX-2 expression. These findings also support our previous studies that showed that dexamethasone delayed rat gastric ulcer healing via inhibition of epithelial cell proliferation in ulcer margins and angiogenesis of ulcer base.1,4 In conclusion, TNF-α increased COX-2 expression and PGE2 formation, and stimulated rat gastric epithelial (RGM-1) cell migration. Dexamethasone


inhibited TNF-α-stimulated RGM-1 cell migration, which was associated with a decrease in COX-2 expression and PGE2 formation.

Acknowledgments This study was supported by grants from the National Science Council of Taiwan (NSC 97-2314-B-075032). The authors thank Miss P.C. Lee (Department of Medicine, Taipei Veterans General Hospital) for her help in editing the figures, and the Clinical Research Core Laboratory, Department of Medical Research and Education, Taipei Veterans General Hospital, for their support.

References 1. Luo JC, Shin VY, Liu ESL, So WHL, Ye YN, Chang FY, Cho CH. Non-ulcerogenic dose of dexamethasone delays gastric ulcer healing in rats. J Pharmacol Exp Ther 2003;307:692–8. 2. Yoo J, Lotz MM, Matthews JB. Cytokines in restitution. In: Cho CH, Wang JY, eds. Gastrointestinal Mucosal Repair and Experimental Therapeutics, 1st edition. Basel: Karger, 2002:14–28. 3. Silen W, Ito S. Mechanisms for rapid re-epithelialization of gastric mucosal surface. Annu Rev Physiol 1985;47:217–29. 4. Luo JC, Shin VY, Liu ESL, Ye YN, Wu WKK, So WHL, Chang FY, et al. Dexamethasone delays ulcer healing by inhibition of angiogenesis in rat stomachs. Eur J Pharmacol 2004;485:275–81. 5. Szabo S, Khomenko T, Gombos Z, Deng XM, Jadus MR, Yoshida M. Review article: transcription factors and growth factors in ulcer healing. Aliment Pharmacol Ther 2000; 14(Suppl):33–43. 6. Tarnawski A, Szabo IL, Husain SS, Soreghan B. Regeneration of gastric mucosa during ulcer healing is triggered by growth factors and signal transduction pathways. J Physio Paris 2001; 95:337–44. 7. Tarnawski AS. Cellular and molecular mechanisms of gastrointestinal ulcer healing. Dig Dis Sci 2005;50(Suppl):S24–33. 8. Basson MD, Modlin IM, Madri JA. Human enterocyte (Caco-2) migration is modulated in vitro by extracellular matrix composition and epidermal growth factor. J Clin Invest 1992;90: 15–23. 9. Martin P. Wound healing—aiming for perfect skin regeneration. Science 1997;276:75–81. 10. Pai R, Szabo IL, Giap AQ, Kawanaka H, Tarnawski AS. Nonsteroidal anti-inflammatory drugs inhibit re-epithelialization of wounded gastric monolayers by interfering with actin, Src, FAK, and tensin signal. Life Sci 2001;69:3055–71. 11. Takahashi S, Shigeta JI, Inoue H, Tanabe T, Okabe S. Localization of cyclooxygenase-2 and regulation of its mRNA expression in gastric ulcers in rats. Am J Physiol Gastrointest Liver Physiol 1998;275:G1137–45. 12. Takahashi M, Ota S, Hata Y, Mikami Y, Azuma N, Nakamura T, Terano A, et al. Hepatocyte growth factor as a key to modulate anti-ulcer action of prostaglandins in stomach. J Clin Invest 1996;98:2604–11. 13. Luo JC, Shin VY, Yang YH, Wu WKK, Ye YN, So WHL, Chang FY, et al. Tumor necrosis factor-α stimulates gastric epithelial cell proliferation. Am J Physiol Gastrointest Liver Physiol 2005;288:G32–8.

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14. Kobayashi I, Kawano S, Tsuji S, Matsui H, Nakama A, Sawaoka H, Masuda E, et al. RGM1, a cell line derived from normal gastric mucosa of rat. In Vitro Cell Dev Biol Animal 1996;32:259–61. 15. Luo JC, Lin HY, Lu CL, Wang LY, Chang FY, Lin HC, Huang YC, et al. Dexamethasone inhibits basic fibroblast growth factor-stimulated gastric epithelial cell proliferation. Biochem Pharmacol 2008;76:841–9. 16. Shin VY, Liu ESL, Koo MWL, Luo JC, So WHL, Cho CH. Nicotine suppresses gastric wound repair via the inhibition of polyamine and K+ channel expression. Eur J Pharmacol 2002; 444:115–21. 17. Netzer P, Halter F, Ma TY, Hoa N, Nguyen N, Nakamura T, Tarnawski A. Interactions of hepatocyte growth factor and non-steroidal anti-inflammatory frogs during gastric epithelial wound healing. Digestion 2003;67:118–28.

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18. Ng KM, Cho CH, Chang FY, Luo JC, Lin HC, Lin HY, Chi CW, et al. Omeprazole promotes gastric epithelial cell migration. J Pharm Pharmacol 2008;60;655–60. 19. Horie-Sakata K, Shimada T, Hiraishi H, Terano A. Role of cyclooxygenase-2 in hepatocyte growth factor-mediated gastric epithelial restitution. J Clin Gastroenterol 1998;27(Suppl): S40–60. 20. Loew D, Schuster O, Graul EH. Dose-dependent pharmacokinetics of dexamethasone. Eur J Clin Pharmacol 1986;30:225–30. 21. Czock D, Keller F, Rasche FM, Haussler U. Pharmacokinetics and pharmacodynamics of systemically administered glucocorticoids. Clin Pharmacokinet 2005;44:61–98. 22. Luo JC, Chi CW, Lin HY, Chang FY, Lu CL, Chen CY, Lee SD. Dexamethasone inhibits epidermal growth factorstimulated gastric epithelial cell proliferation. J Pharmacol Exp Ther 2007;320:687–94.
